Bruno Casetta

Quantitative analysis of bile acids in human plasma by liquid chromatography-electrospray tandem mass spectrometry: a simple and rapid one-step method.

Abstract Bile acids play a pivotal role in the metabolism of cholesterol and lipids. Their blood concentrations are important prognostic and diagnostic indicators of hepatobiliary and intestinal dysfunction. This class of molecules comprises a heterogeneous group of compounds with a common cholesterol scaffold. Recently, the introduction of liquid chromatography coupled to tandem mass spectrometry methods has revealed an innovative path in the quantisation of specific bile acids in biological specimens. A robust and sensitive method has been developed based on high performance liquid chromatography separation coupled to an electrospray triple-quadrupole mass spectrometer. Human plasma samples were analysed on a C18 reverse-phase column. The elution profiles were monitored in multiple reaction-monitoring mode, quantifying and identifying each analyte by its own unique precursor to product patterns. A linear correlation over a broad range of bile acid concentrations (0.1–100 μM) was observed. The average recovery period for all of the analysed bile acids was 98±3%. Intra-day and inter-day precision averages were 2% and 5.4%, respectively. The determination was achieved within a single chromatographic run for all unconjugated, glycine-and taurine-conjugated isomeric forms of bile acids. As a proof of principle this method has been validated on a small subset of cholestatic patients (n = 7) and compared to appropriate clinical controls (n = 10). Based upon our encouraging experimental results, the described HPLC separation coupled to tandem mass spectrometry method for the analysis of bile acids in biological samples is deemed a robust and accurate procedure. Consequently, we propose this technique as a suitable candidate method for the identification and quantitation of bile acids in routine analysis.

Second-tier test for quantification of alloisoleucine and branched-chain amino acids in dried blood spots to improve newborn screening for maple syrup urine disease (MSUD).

BACKGROUND Newborn screening for maple syrup urine disease (MSUD) relies on finding increased concentrations of the branched-chain amino acids (BCAAs) leucine, isoleucine, and valine by tandem mass spectrometry (MS/MS). d-Alloisoleucine (allo-Ile) is the only pathognomonic marker of MSUD, but it cannot be identified by existing screening methods because it is not differentiated from isobaric amino acids. Furthermore, newborns receiving total parenteral nutrition often have increased concentrations of BCAAs. To improve the specificity of newborn screening for MSUD and to reduce the number of diet-related false-positive results, we developed a LC-MS/MS method for quantifying allo-Ile. METHODS Allo-Ile and other BCAAs were extracted from a 3/16-inch dried blood spot punch with methanol/H2O, dried under nitrogen, and reconstituted into mobile phase. Quantitative LC-MS/MS analysis of allo-Ile, its isomers, and isotopically labeled internal standards was achieved within 15 min. To determine a reference interval for BCAAs including allo-Ile, we analyzed 541 dried blood spots. We also measured allo-Ile in blinded samples from 16 MSUD patients and 21 controls and compared results to an HPLC method. RESULTS Intra- and interassay imprecision (mean CVs) for allo-Ile, leucine, isoleucine, and valine ranged from 1.8% to 7.4%, and recovery ranged from 91% to 129%. All 16 MSUD patients were correctly identified. CONCLUSIONS The LC-MS/MS method can reliably measure allo-Ile in dried blood spots for the diagnosis of MSUD. Applied to newborn screening as a second-tier test, it will reduce false-positive results, which produce family anxiety and increase follow-up costs. The assay also appears suitable for use in monitoring treatment of MSUD patients.

Development of a highly sensitive method for the quantification of estrone and estradiol in serum by liquid chromatography tandem mass spectrometry without derivatization

Measurement of estrone (E1) and estradiol (E2) values <1 pg/mL (3.7 pmol/L) is necessary for postmenopausal, pediatric and male serum samples. Until now this was rarely reached and only through derivatization which can present problems for estradiol. A very sensitive LC-MS/MS method was developed avoiding derivatization, convenient for large-scale studies. The desired sensitivity and specificity were achieved using ESI negative mode, LLE and a 2D chromatography consisting of a trapping column and a second dimension reverse-phase C8 analytical column. A mixture of an aqueous solution of ammonium fluoride at 0.2mM and methanol was used on the analytical column to further increase the sensitivity. Serum LOQ was <0.5 pg/mL (1.9 pmol/L) for E2 and E1 and recoveries ranged from 95 to 105%. No carry-over was detectable. Inter assay CVs were 4.0% at 21 pg/mL (77 pmol/L) for E2, 7.6% at 25 pg/mL (93 pmol/L) for E1. Comparison with commercial direct estrogen assays (Roche Diagnostics E170 for E2, Bioline RIA for E1) exposed analytical unsuitability (due to a combined lack of sensitivity and specificity) for the assay of male, postmenopausal or pediatric samples.

Development of a method for rapid quantitation of amino acids by liquid chromatography-tandem mass spectrometry (LC-MSMS) in plasma.

Abstract A new analytical method has been developed and is proposed for the rapid determination of eighteen common amino acids, including tryptophan, in plasma and dried blood spots, by liquid chromatography coupled with ionspray tandem mass spectrometry. Potentially the method can include other amino acids and can be used for the diagnosis of metabolic disease. The use of the ionspray tandem mass spectrometry approach permits extremely rapid chromatographic separation of all amino acids requiring less than four minutes for the analysis of each sample, after a simple sample preparation procedure. The chromatographic separation of the analytes was achieved using a CN normal phase column and a water/acetonitrile/trifluoroacetic acid mobile phase at flow rate of 1 ml/min. The mass spectrometer was operated in multiple reaction monitoring mode, where each analyte had its own unique precursor and product ion setting. The quantitative analysis of amino acids was achieved using as internal standards just two representative isotopically labeled amino acids: D4-Ala and D5-Phe. Calibration is made externally by using aqueous solutions with the same labelled amino acids as internal standards. The high specificity of tandem mass spectrometry coupled with a fast chromatographic process is suitable for the rapid and reliable assay of metabolically significant amino acids. The liquid chromatography-tandem mass spectrometry method is more effective than other published tandem mass spectrometry methods at distinguishing isobaric amino acids like Leu, Ile and HO-Pro and certainly far more rapid than HPLC or ion-exchange chromatographic methods.

Development of a method for the quantification of 1α,25(OH) 2 –vitamin D 3 in serum by liquid chromatography tandem mass spectrometry without derivatization

Quantification of 1α,25(OH)2–vitamin D3 in serum is technically challenging because of its very low concentration. Even mass spectrometry faces a challenge due to the low sensitivity for this molecule, unless a specific derivatization is implemented. Therefore, there is still a need for a robust, simplified, sensitive and specific liquid chromatography tandem mass spectrometry (LC-MS/MS) method for the quantification of 1α,25(OH)2–vitamin D3. Thanks to tandem mass-spectrometry associated to an on-line sample extraction and clean-up, sample preparation has been reduced to a simple protein precipitation step and derivatization has been avoided. Specimen volume has been kept to a minimum. The innovative aspects of the hereby-presented method are the use of stable lithium adducts and a highly sensitive tandem mass spectrometer. The achieved limit of quantification is at a level of 15 pg mL−1, with a % CV of 5–15% at physiological concentration levels. The linearity is good and spiking experiments yielded a recovery of 87–102%. Comparison of selected samples with an established reference method, using an extensive purification procedure (high-performance liquid chromatography and radioimmunoassay), has shown a good correlation.

A robust liquid chromatography tandem mass spectrometry method for total plasma homocysteine determination in clinical practice.

Abstract Background: Total plasma homocysteine has emerged as an independent risk factor for vascular disease. To meet increasing requests by clinicians for this homocysteine determination, a rapid assay for a routine use has been developed. Methods: A robust, stable, isotope-dilution liquid chromatography tandem mass spectrometry (LC/MS/MS) method is described, including all the practical details and analytical performance results. Results: The method allows homocysteine quantitation over a linear working range up to 100μmol/L, with the limit of quantification estimated at a low value of 0.09μmol/L. Total analytical imprecision is less than 4%. Accuracy was assessed by measuring the homocysteine concentration in a serum Standard Reference Material. Conclusions: The method was demonstrated to be quick, reliable and cheap after 1year of use on a time-shared instrument in our hospital unit. Clin Chem Lab Med 2006;44:987–90.

Characterization of cisplatin-glutathione adducts by liquid chromatography-mass spectrometry evidence for their formation in vitro but not in vivo after concomitant administration of cisplatin and glutathione to rats and cancer patients

After incubation of equimolar amounts of cisplatin (CDDP) and glutathione (GSH) in phosphate buffer pH 7.4 at 37 degrees C, we detected two CDDP-GSH adducts whose structures, characterized by LC-MS, corresponded to cis-[Pt(NH3)2Cl(SG)] and cis-([Pt(NH3)2Cl]2(mu-SG))+. The latter is a new CDDP-GSH adduct, which was postulated but never structurally characterized so far. Rats and patients were given a 15-min intravenous infusion of CDDP (10 mg/kg to rats and 25 mg/m2 to patients) preceded by a GSH intravenous administration (200 mg/kg to rats as a bolus and 1.5 g/m2 to patients as a 15-min infusion). After the administrations, CDDP-GSH adducts were absent in rat and human plasma ultrafiltrates. The discrepancy between in vitro and in vivo findings can be explained based on pharmacokinetic considerations.

Study of β-cyclodextrin-ketoconazole-tartaric acid multicomponent non-covalent association by positive and negative ionspray mass spectrometry

In continuation of studies of multicomponent non-covalent associations (MCAs) of cyclodextrin (CD) inclusion or host–guest (H–G) complexes of hydrophobic or barely water-soluble drugs with suitable counter ions, which can dramatically increase the hydrosolubility of the guest drug, was the β-CD–KC–tartaric acid (TA) MCA, where KC=ketoconazole, an antifungal drug, investigated by ionspray (IS) mass spectrometry (MS) and MS/MS in both the positive and negative ion modes. In the positive IS mode a protonated 1:1:1 β-CD–KC–TA gaseous species is obtained, which dissociates by the loss of TA upon collisional activation (CA), thus reproducing the same behaviour as observed previously for a β-CD–terfenadine–TA MCA. Unprecedented results were obtained in the negative ion mode. In particular, deprotonated 1:1:1 β-CD–KC–TA MCA was detected, which upon CA yielded mainly deprotonated 1:1 β-CD–TA and tartrate anion. Hence, while a relatively strong interaction binding β-CD to TA within the MCA parent anion emerges, the fair abundance of tartrate anion could suggest the formation of its neutral complementary fragment, 1:1 β-CD–KC, a possibly H–G complex not observed as a negatively charged MS/MS fragmentation product. The role of the KC–TA ionic bonding of the neutral MCA appears very pertinent to the study by positive and negative ISMS of the non-covalent interactions within the gaseous protonated or deprotonated ternary complex thereof.

High-throughput plasma docetaxel quantification by liquid chromatography-tandem mass spectrometry.

BACKGROUND The most valuable treatment option for breast, prostate and lung carcinomas is at present represented by a low dose of docetaxel, administered on a weekly basis. A better understanding of docetaxel pharmacokinetic and pharmacodynamic profiles could lead to an improvement in this dose regimen efficacy. METHODS In this study a high-throughput method is described for the rapid quantification of docetaxel for large clinical pharmacology investigations. This analytical approach is based on an automatic on-line purification and enrichment technique followed by a measurement in tandem mass spectrometry through Multiple Reaction Monitoring. RESULTS The assay was validated over a 0.15-1500 ng/mL range. Intra-day precision ranged from 1.9% to 6.4%, while the inter-day was between 7.6% and 11.2%. The mean deviation from the nominal value ranged from -0.5% to 5.6% for the intra-day, and from -0.4% to 3.1% for the inter-day assay. Clinical applicability was demonstrated by measuring plasma pharmacokinetics in patients receiving weekly 25-35 mg/m(2) of docetaxel. CONCLUSION The proposed LC-MS/MS assay was found to have a better performance than previously reported methods in terms of sensitivity and sample preparation. It does not require any laborious pre-analytical manipulation and can be easily employed in large clinical pharmacology studies.

Sulfamethazine, Sulfothiazole and Albendazole Residue Dosage in Food Products Determined by Liquid Chromatography/Tandem Mass Spectrometry

There is a growing demand for analytical techniques for the detection of a wide variety of residues from synthetic molecules in matrices such as soil, water, air and food. These techniques have to meet the requirements of speed and sensitivity as well as the ability to handle any matrix with minimal sample clean-up. Features of mass spectrometry combined with liquid chromatography can fulfill these requirements as is shown by this work which reports the use of ion spray ionization coupled with tandem mass spectrometry for the detection of some drug residues. In particular, the direct use of existing LC methods, originally conceived for use with some other sort of detector, is demonstrated.